Metabolic Profiling of Colorectal Adenocarcinoma Using Mass Spectrometry Imaging

Kolorektalni karcinom čini više od 90 % malignih tumora debelog crijeva, a učestalost ove bolesti u postupnom je porastu. Najrašireniji oblik je adenokarcinom, koji je, između ostalog, rezultat složenih metaboličkih interakcija u stanicama karcinoma na koje utječe i mikrookoliš. Biokemijska svojstva zloćudnih stanica među pacijentima značajno se razlikuju, a putem konvencionalnih histopatoloških pristupa teško ih je detaljnije opisati i klasificirati. Posljedica toga jednaki su terapijski postupci u bolesnika s različitom molekularnom podlogom bolesti. Stoga, cilj ovog istraživanja bio je vizualizirati i identificirati specifične endogene metabolite kolorektalnog adenokarcinoma i susjednog peritumorskog tkiva upotrebom slikovne spektrometrije masa (MSI od eng. Mass Spectrometry Imaging) kod deset ispitanika. MSI je izvedena primjenom matricom potpomognute ionizacije laserskom desorpcijom s analizatorom vremena preleta (MALDI-TOF od eng. Matrix-Assisted Laser Desorption/Ionization-Time Of Flight) u pozitivnom i negativnom načinu snimanja. Dobiveni rezultati MSI tehnikom pokazali su jasnije razlikovanje morfoloških obrazaca tumorskog i peritumorskog tkiva u odnosu na nativni i hematoksilin-eozin (H&E) metodom obojani preparat. Slikovna analiza na temelju m/z signala dobivenih snimanjem u negativnom načinu bolje je razlikovala vrstu tkiva i stanica te pružila informativnije raspodjele iona metabolita u odnosu na pozitivan način snimanja. Analiza glavnih komponenti (PCA od eng. Principal Component Analysis) jasno je klasificirala tumorske i peritumorske regije za pojedine ispitanike na temelju razlika u njihovim m/z signalima. Uočeno je da molekularne komponente većih masa bolje razlikuju navedene regije. Primjenom t-testa nije identificiran statistički značajan m/z signal svojstven tkivima svih ispitanika, što ukazuje na heterogenost tumorskih i peritumorskih regija među svim uzorcima. Kao dominantni spojevi u obje vrste tkiva detektirani su ceramidi, što bi moglo ukazati da su promjene u metabolizmu ceramida usko povezane s nastankom i napredovanjem kolorektalnog adenokarcinoma. Ovo istraživanje potvrđuje da nema univerzalnih markera adenokarcinoma kolona iz grupe malih molekula te upućuje na potrebu za personalizacijom dijagnostike i, shodno tome, terapijskog pristupa kolorektalnom adenokarcinomu.Colorectal carcinoma represents more than 90 % of malignant tumors of the colon, and the incidence of this disease is gradually increasing. The most common form is adenocarcinoma, which is, among other things, the result of complex metabolic interactions in cancer cells that are also affected by the microenvironment. The biochemical properties of malignant cells differ significantly among patients, and it is hard to describe and classify them in more detail through conventional histopathological approaches. The consequence is equal therapeutic procedures in patients with different molecular background of diseases. Therefore, the aim of this study was to visualize and identify specific endogenous metabolites of colorectal adenocarcinoma and surrounding peritumoral tissue using Mass Spectrometry Imaging (MSI) in ten patients. MSI was performed using Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) in positive and negative mode. The results obtained by the MSI technique showed a clearer difference between the morphological patterns of the tumor and peritumoral tissue in relation to the native preparation and preparation stained by hematoxylin-eosin (H&E) method. Image analysis based on m/z signals obtained by negative ion mode better distinguished tissue and cell type and provided a more informative distribution of ionized metabolites compared to positive ion mode. Principal component analysis (PCA) clearly classified tumor and peritumor areas based on differences in their m/z signals in the individual sample analysis. It has been observed that the molecular components of higher masses better differentiate these areas. No common statistically significant m/z signal was found in all subjects using the t-test, indicating heterogeneity of tumor and peritumor regions among all samples. Ceramides have been identified as dominant compounds in both tissue types, which may mean that changes in ceramide metabolism are closely related to the development and progression of colorectal adenocarcinoma. This study confirms that there are no universal tumor markers from the group of small molecules and requisit the need for personalization of diagnostics and, consequently, therapeutic approach to colorectal adenocarcinoma